Biomarker changes in suspected idiopathic normal-pressure hydrocephalus patients undergoing external lumbar drainage: a pilot study

Aim To examine whether changes in biomarker concentrations in patients with idiopathic normal-pressure hydrocephalus (iNPH) during 72 h of external lumbar drainage (ELD) can differentiate between responders and non-responders. Methods Twenty patients with clinical and neuroradiological signs of iNPH underwent ELD over a period of 72 h. During this period, changes in cerebrospinal fluid (CSF) concentrations of biomarkers (amyloid-β, total and phosphorylated tau proteins) and intracranial pressure were monitored, and the volume of drained CSF was measured. Changes in the concentrations of selected biomarkers at three time points (0, 36, and 72 h) during ELD were tested for association with changes in clinical condition. Results Ten patients showed significant clinical improvement after ELD, quantified as a difference of two or more points on the Mini-Mental State Examination and/or Japanese iNPH grading scale. The concentration of all tested biomarkers increased during the first 36 h. Respondents had higher Aβ 1-42 at all time points, with a significant difference seen after 72 h. They also had a significantly higher Aβ1-42/Aβ1-40 ratio at all time points. Conclusion A gradual increase in Aβ 1-42 concentration during three-day ELD represents a possible positive prognostic factor for the placement of permanent CSF drainage in patients with iNPH.

The most common differential diagnosis for iNPH is Alzheimer disease (AD), the most prevalent neurodegenerative condition (12)(13)(14).Up to 75% of patients exhibit pathohistological characteristics of both AD and iNPH (14)(15)(16)(17)(18).For AD diagnosis, three core biomarkers are crucial: amyloid beta 1-42 (Aβ 1-42), total tau (t-tau), and phosphorylated tau protein (p-tau) (19,20).The full-length Aβ 1-42 is extremely hydrophobic and forms oligomers and fibrils that accumulate in extracellular plaques, which are characteristic of AD (17).Presumably due to the accumulation of Aβ 1-42 in plaques, its concentration in cerebrospinal fluid (CSF) is notably lower in AD patients than in the healthy population (21)(22)(23)(24)(25)(26).Although in patients with iNPH, amyloid-β concentration tends to be lower than in the healthy population, these values are still higher than those in patients with AD (27).Patients with iNPH have a higher concentration of total tau protein (28), p-tau protein, and amyloid precursor protein (APP) and its fragments in CSF than the healthy population, but lower than AD patients (29).Another useful marker in discriminating iNPH from AD may be CSF phosphorylated tau protein at threonine position 181 (pT181), alone or in combination with total tau (30,31).
Factors determining CSF movement along the CSF system, which affect its interaction with interstitial fluid, certainly influence the fate of molecules of different molecular weight and their distribution between tissue and CSF (32)(33)(34).Healthy people, for instance, have a higher concentration of monoamines and their metabolites in cranial than in lumbar CSF, a finding that challenges the traditional view of a unidirectional CSF circulation (35,36).This suggests a more complex interaction between CSF and brain tissue metabolism.When 10 mL (37) to 15 mL (38) of lumbar CSF was sampled, the concentration of various monoamines exponentially increased in individual fractions of the CSF sample (first, middle, and last milliliters).This observation indicates that CSF closer to the brain tissue contains a higher concentration of monoamines and their metabolites, contrary to an even distribution expected according to the classic concept of one-way CSF circulation, reflecting changes in the metabolism of adjacent tissue (37).In addition, in CSF samples obtained by free cisternal drainage in animals over two hours, the concentration of monoamine metabolites exponentially increased, which indicated an influx of CSF from higher parts containing higher concentrations of the measured substances (39).
Contrary to the concentrations of monoamines within the CSF system, the concentrations of blood-derived proteins in the ventricle are lower compared with the lumbar compartment (40,41).When extracting CSF through lumbar puncture in healthy people, the protein concentration in CSF decreases in subsequent fractions (37).This observation additionally indicates that CSF is not mixed by circulation.Moreover, extracting a CSF sample from higher parts with a lower protein concentration can easily explain the observed phenomenon of protein concentration drop in lumbar CSF in later fractions.
iNPH is characterized by biochemical changes in CSF that reflect metabolic changes in the brain.CSF is in direct contact with the extracellular space and is therefore considered a good source of potential biomarkers.Similar to the concentration gradient for proteins in the CSF system, there also appears to be a concentration gradient for peptides such as Aβ 1-42 (42).The preoperative concentration of Aβ 1-42 in the lumbar region was shown to be higher than the postoperative concentration in the ventricles (42).
In contrast to healthy people, patients with AD and iNPH are expected to have lower Aβ 1-42 levels in initial CSF samples due to amyloid accumulation in the interstitial space.Specifically, during preoperative testing, patients with suspected iNPH undergoing prolonged external lumbar drainage (ELD) with larger CSF volumes drained, are expected to have an increased peptide biomarker concentration.This is because low CSF pressure may induce hydrostatic drawing of water from the blood into the interstitial space and CSF, resulting in the "washing out" of accumulated Aβ 1-42 (43,44).We hypothesized that changes in peptide concentration in the CSF of patients evaluated for potential iNPH management differed from those observed previously in healthy people.Furthermore, we postulated that Aβ concentration from the onset to the end of prolonged CSF drainage, due to suspected iNPH, varied between responders and non-responders.Consequently, we assessed AD biomarker concentrations in collected CSF samples during extended ELD immediately after placement, and after 36 and 72 hours, while moni-toring changes in CSF pressure, volume of drained CSF, and clinical response.

Patients
This study was conducted at the University Hospital Center Zagreb from February 2018 to April 2023.The research was approved by the Ethics Committee of the University Hospital Center Zagreb and the Ethics Committee of the University of Zagreb School of Medicine.
The study enrolled patients who underwent drainage placement due to suspected iNPH manifesting as cogni-tive impairment, gait disturbances, and urinary incontinence.To quantify cognitive impairment, the Mini-Mental Status Examination (MMSE) was used.The Japanese iNPH scale (JiNPHS) was used to quantify other symptoms.A MMSE score of less than 24 indicated significant cognitive deterioration (45).
Patients were required to meet the neuroradiological criteria of iNPH, including ventriculomegaly, especially of the frontal and temporal horns, Evans index >0.3,bulging of the corpus callosum more cranially, callosal angle <90°, significant expansion of the Sylvian fissure disproportionate to the expansion of the convexity sulcus (especially in the parietal region), and increased signal in T2-measured periventricular MRI sequences.In patients with clinical and radiological sus- picion of iNPH, standard testing involved placing an external lumbar drain and draining a large amount of CSF ( ~ 10 mL/h) over a 72-hour period.The exclusion criteria were underlying neurological or non-neurological disorders that can cause the same symptoms and a history of severe brain trauma.The opening CSF pressure during ELD placement had to be within reference values, meaning ≤20 cm H 2 O. Before drainage placement, patients were thoroughly evaluated for the severity of clinical symptoms (MMSE and Japanese iNPH scale).A follow-up clinical evaluation was performed after 72 hours (Figure 1).Patients who had shunt surgery were followed up at 1-month and 6-month intervals postoperatively.Nine patients remained clinically stable, while one required shunt revision due to infection.

Method of CSF pressure recording during ELd
Lumbar drainage was placed between the intervertebral space L IV /L V or L V /S I , depending on the anatomical predisposition of the patients, and was determined in relation to the iliac crest.The puncture site was prepared in the standard way, and a 14-16 G Tuohy needle was inserted.After removing the stylet, the needle was connected to the monitor via a transducer placed at the level of the internal auditory canal (IAC), and the first lumbar opening pressure was measured.Subsequently, a lumbar drain (Medtronic EDM Lumbar Drainage Kit, Minneapolis, MN, USA) was placed through the needle and connected to the transducer and the CSF collector via the T connection.CSF pressure was monitored hourly in the horizontal position, corresponding to intracranial pressure when the pressure transducer is positioned at the level of the IAC.
Since biomarker concentrations can sometimes vary significantly (46), each sample was analyzed in duplicate.Each biomarker was measured on the same day using the same batch of reagents.If the measured concentrations of a sample differed by more than 10%, the measurement was repeated.All reported concentrations are the mean values of two measurements.

Statistical analysis
Continuous variables are presented as median (range or interquartile range), and nominal variables as absolute (relative) frequency.Differences between the groups in continuous variables were assessed with the Mann-Whitney U test.Differences between the groups in nominal variables were assessed with the χ 2 test.The before-and after-ELD placement comparisons were performed with the Wilcoxon signed-rank test.Multiple comparisons of biomarker levels across time were performed with the Friedman test.All tests were two-sided.The level of statistical significance was set at P = 0.05.SPSS, version 25.0 (IBM Corp., Armonk, NY, USA), and GraphPad Prism, version 8.4.3 (GraphPad Software, Boston, MA, USA), were used for statistical analysis and graphical presentation of the results.Out of all patients tested for suspected iNPH, 10 showed significant clinical improvement after ELD (responders), quantified as a difference of two or more points in either MMSE or JiNPHS.Over three days, the average CSF volume drained was 770 mL, ranging from 570 to 982 mL.In the responder group, an average of 730 mL of CSF was drained over 72 hours, compared with 787 mL in the non-responder group (Table 1).
The average opening pressure during ELD placement was 6.5 cm H 2 O in both groups.After 36 h, the median pressure in the responder group increased to 7.5 cm H 2 O, while it averaged 5 cm H 2 O in the non-responder group.The pressure difference between the groups decreased after 72 h (6 cm H 2 O and 5 cm H 2 O, respectively).
The levels of Aβ1-42, Aβ1-40, total tau, and pT181 biomarkers significantly changed over time, with the exception of the Aβ1-42/Aβ1-40 ratio (Table 3).Aβ 1-42 levels were higher in responders compared with non-responders at all measured points, with a significant difference noted only after 72 h (Figure 2A, Table 4).Although non-responders had higher total tau (Figure 2C) and Aβ 1-40 levels (Figure 2B) at all time points, the differences were not significant.Similarly, pT181 levels were higher in the responder group at all times, but the difference did not reach significance (Figure 2D).Responders had a significantly higher Aβ1-42/ Aβ1-40 ratio at all time points (Figure 2E, Table 4).
diSCuSSion  (20,21).A meta-analysis of 13 studies (48-60) showed that shunt-responder patients had lower lumbar CSF concentrations of total and phosphorylated tau proteins than non-responders, with no significant difference in Aβ 1-42 levels (61).However, the generalizability of these results is limited due to variations in analytical methods, methodological weaknesses of the studies, the small number of studies dealing with Aβ 1-42 changes, and differing CSF sample collection times after varying drainage durations.Tarnaris et al demonstrated, in 11 patients with suspected iNPH, that the concentrations of Aβ1-42 and total tau protein increased during 72 h of CSF evacuation via ELD (62).
Jingami et al found that during the tap test, there was an increased concentration of total tau protein and decreased levels of Aβ 1-42 in the last milliliter compared with the first milliliter of CSF (63).Considering that the volume of drained CSF in the cited study was 30 mL, these results cannot be directly compared with ours, where the drained CSF volume was considerably higher (770 mL).
Our findings demonstrate changes at three time points (0' , 36 h, 72 h) during ELD of a significant total CSF volume (average 770 mL under lower CSF pressure).In the responder group, the concentration of the Aβ 1-42 isoform was consistently higher at all time points compared with the nonresponder group, with the difference reaching significance only after 72 h.This observation, under these specific conditions, significantly differs from those of previous studies.Additionally, in our study, the non-responder group had higher total tau protein and Aβ 1-40 at all time points, though without reaching statistical significance.In the responder group, the concentration of total tau protein decreased after 72 h, which indicated that following the initial washout of this marker characteristic of neurofibrillary neurodegeneration, its concentration in patients without AD comorbidity in the lumbar CSF sample stabilizes.Higher values of pT181 were also observed in the responder group at all time points, but without a significant difference, which contrasts with the findings of previous studies, in which responders had lower values of pT181.
Another longitudinal study, tracking the concentrations of biomarkers in the lumbar and ventricular spaces over time, showed that in patients with probable iNPH and a negative Aβ brain biopsy, the concentration of Aβ 1-42 shifted toward more positive values compared with patients with iNPH and a positive Aβ brain biopsy, particularly after a longer follow-up.This indicates that, in patients with probable iNPH, a higher Aβ 1-42 value acts as the best negative predictor of underlying AD (64).These findings support the notion of a better clinical response to permanent shunt placement in patients with higher Aβ 1-42 levels in the lumbar CSF, aligning well with our results.
According to the new concept of CSF physiology, CSF can be produced at the level of the brain and spinal tissue capillary network if an osmotic or hydrostatic gradient is created, which facilitates the entry of a net volume of fluid from the capillaries into the interstitial space and CSF (43,44).During ELD, the reduction of hydrostatic CSF pressure to 5-7.5 cm H 2 O enables the entry of fluid from the capillary network into the interstitial space of the cerebral gray matter and potentially facilitates the "washout" of substances accumulated in the interstitium into the CSF system.This mechanism may explain the observed gradual increase in Aβ 1-42 concentration during prolonged ELD in responders.
The current study is subject to several major limitations, including a small sample size, single-center design, and the absence of long-term follow-up data.These factors limit the generalizability of our findings and the ability to assess how biomarker changes correlate with long-term outcomes, such as the progression of neurological symptoms and mortality.Additionally, the lack of a control group to directly compare the biomarker levels may also affect the interpretation of our results.Without a control group, it is challenging to ascertain whether the observed biomarker  declaration of authorship KBM, GM, MK conceived and designed the study; KBM, HB, SMM acquired the data; KBM, GM, HB, GŠ, EŠP, MK analyzed and interpreted the data; KBM, MK drafted the manuscript; all authors critically reviewed the manuscript for important intellectual content; all authors gave approval of the version to be submitted; all authors agree to be accountable for all aspects of the work.
Competing interests HB is a Deputy Editor-in-Chief of the Croatian Medical Journal.To ensure that any possible conflict of interest relevant to the journal has been addressed, this article was reviewed according to best practice guidelines of international editorial organizations.All authors have completed the Unified Competing Interest form at www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.

taBLE 1 .
Sociodemographic and clinical data for responder and non-responder groups.numbers are median (range) or absolute *abbreviations: MMSE -Mini-Mental State Examination; JinPHS -Japanese inPH Scale; ELd -external lumbar drainage, CSF -cerebrospinal fluid.Croat Med J.
our study, the concentration of all tested biomarkers increased within the first 36 hours after drainage placement.
Funding This work was supported by the Croatian Science Foundation and the Ministry of Science and Education of the Republic of Croatia (Projects: 1. Pathophysiology of Cerebrospinal Fluid and Intracranial Pressure.No. 108-1080231-0023; 2. Cerebrospinal Fluid Volume and Pressure Regulation.No. 10106-20-2502; 3. Role of Blood-Brain Barrier, Innate Immunity, and Tau Protein Oligomerization in the Pathogenesis of Alzheimer's Disease." No. CSF IP-2019-04-3584).Research was co-financed by the Scientific Centre of Excellence for Basic, Clinical, and Translational Neuroscience CORE-NEURO (project Experimental and Clinical Research of Hypoxic-Ischemic Damage in Perinatal and Adult Brain; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund).Ethical approval granted by the the Ethics Committee of the Clinical Hospital Center Zagreb (Class: 8.1-17/202-2, number: 02/21 AG), and by the Ethics Committee of the University of Zagreb School of Medicine (Class: 641-01/23-02/01, number: 251-59-10106-23-111/213).